U.S. patent number 11,389,492 [Application Number 15/558,669] was granted by the patent office on 2022-07-19 for method for inducing oral tolerance via administration of beta-lactoglobulin derived peptides in combination with probiotic.
This patent grant is currently assigned to N.V. NUTRICIA. The grantee listed for this patent is N.V. Nutricia. Invention is credited to Johan Garssen, Leon Matthieu Johannes Knippels.
United States Patent |
11,389,492 |
Knippels , et al. |
July 19, 2022 |
Method for inducing oral tolerance via administration of
beta-lactoglobulin derived peptides in combination with
probiotic
Abstract
The invention pertains to the use of a probiotic and a
beta-lactoglobulin-derived peptide in the manufacture of a product
for use in inducing oral tolerance, and/or treatment, prevention or
reducing the risk of allergy in a subject, in particular cow's milk
protein allergy.
Inventors: |
Knippels; Leon Matthieu
Johannes (Utrecht, NL), Garssen; Johan (Utrecht,
NL) |
Applicant: |
Name |
City |
State |
Country |
Type |
N.V. Nutricia |
Zoetermeer |
N/A |
NL |
|
|
Assignee: |
N.V. NUTRICIA (Zoetermeer,
NL)
|
Family
ID: |
1000006441973 |
Appl.
No.: |
15/558,669 |
Filed: |
March 17, 2016 |
PCT
Filed: |
March 17, 2016 |
PCT No.: |
PCT/NL2016/050190 |
371(c)(1),(2),(4) Date: |
September 15, 2017 |
PCT
Pub. No.: |
WO2016/148572 |
PCT
Pub. Date: |
September 22, 2016 |
Prior Publication Data
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|
|
|
Document
Identifier |
Publication Date |
|
US 20180110811 A1 |
Apr 26, 2018 |
|
Foreign Application Priority Data
|
|
|
|
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Mar 18, 2015 [NL] |
|
|
PCT/NL2015/050173 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L
33/18 (20160801); A61K 38/1722 (20130101); A23L
33/135 (20160801); A61K 35/744 (20130101); A61K
35/745 (20130101); A23L 33/40 (20160801); A61K
38/018 (20130101); A61K 31/702 (20130101); A23L
33/30 (20160801); A23L 33/19 (20160801); A61P
37/08 (20180101); A61K 45/06 (20130101); A61K
31/702 (20130101); A61K 2300/00 (20130101); A23V
2002/00 (20130101); A23Y 2300/29 (20130101); A23Y
2300/55 (20130101); A23V 2002/00 (20130101); A23V
2200/304 (20130101); A23V 2200/3204 (20130101); A23V
2250/54244 (20130101); A23V 2250/55 (20130101) |
Current International
Class: |
A01N
63/00 (20200101); A23L 33/19 (20160101); A23L
33/18 (20160101); A23L 33/00 (20160101); A61K
38/17 (20060101); A61P 37/08 (20060101); A61K
45/06 (20060101); A23L 33/135 (20160101); A61K
31/702 (20060101); A61K 35/744 (20150101); A61K
38/01 (20060101); A61K 35/745 (20150101) |
Field of
Search: |
;424/93,400 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
|
|
|
2013407963 |
|
Oct 2019 |
|
AU |
|
1 364 586 |
|
Nov 2003 |
|
EP |
|
2 436 389 |
|
Apr 2012 |
|
EP |
|
2008-195618 |
|
Aug 2008 |
|
JP |
|
2014/0030354 |
|
Mar 2014 |
|
KR |
|
WO-02/24883 |
|
Mar 2002 |
|
WO |
|
WO-2011/151060 |
|
Dec 2011 |
|
WO |
|
WO-2013/083691 |
|
Jun 2013 |
|
WO |
|
Other References
Prescott et al. Journal of Allergy and Immunology , vol. 120, issue
2, pp. 255-262, 2007 (Year: 2007). cited by examiner .
Prioult et al. Clinical and Diagnostic Laboratory Bacteriology vol.
10, No. 5, pp. 787-792 , Sep. 2003 (Year: 2003). cited by examiner
.
Guenolee et al., "Stimulation of interleukin-10 production by
acidic beta-lactoglobulin-derived peptides hydrolyzed with
lactobacillus paracasei NCC2461 peptidases", Clinical and
Diagnostic Laboratory Immunology, Mar. 2004, vol. 11, No. 2, pp.
266-271. cited by applicant .
International Search Report issued in International Patent
Application No. PCT/NL2016/050190, dated Jul. 18, 2016. cited by
applicant.
|
Primary Examiner: Hines; Jana A
Assistant Examiner: Shahnan Shah; Khatol S
Attorney, Agent or Firm: Foley & Lardner LLP
Claims
The invention claimed is:
1. A method for inducing oral tolerance, and/or treating, or
reducing the risk of allergy in a subject, comprising administering
to the subject a composition comprising: (i) 10.sup.2-10.sup.13
cfu, per g dry weight of the composition, of a probiotic
Bifidobacteria breve, and (ii) 2 to 10 distinct
beta-lactoglobulin-derived peptides comprising an amino acid
sequence consisting of 14 to 25 consecutive amino acids from amino
acid nos. 13 to 48 of the beta-lactoglobulin protein represented by
SEQ ID No. 1.
2. The method according to claim 1, wherein the
beta-lactoglobulin-derived peptide consists of an amino acid
sequence selected from the group consisting of SEQ ID Nos. 2-5,
optionally coupled to 1-6 further amino acids at its C- and/or
N-terminus.
3. The method according to claim 1, wherein the composition further
comprises a prebiotic.
4. The method according to claim 3, wherein the prebiotic is
selected from the group consisting of fructo-oligosaccharide,
non-digestible dextrin, galacto-oligosaccharide,
xylo-oligosaccharide, arabino-oligosaccharide,
arabino-galacto-oligosaccharide, gluco-oligosaccharide,
glucomanno-oligosaccharide, galactomanno-oligosaccharide,
mannan-oligosaccharide, chito-oligosaccharide, uronic acid
oligosaccharide, sialyl-oligosaccharide and
fuco-oligosaccharide.
5. The method according to claim 3, wherein the prebiotic comprises
a galacto-oligosaccharide and/or a fructo-oligosaccharide.
6. The method according to claim 4, wherein the prebiotic comprises
a mixture of a short-chain oligosaccharide having an average degree
of polymerisation of 2-8 and a long-chain oligosaccharide having an
average degree of polymerisation of 10-60.
7. The method according to claim 1, comprising a further probiotic
strain selected from the group consisting of Lactobacillus
acidophilus, Lactobacillus paracasei, Lactobacillus johnsonii,
Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus
rhamnosus, Lactobacillus casei, Lactobacillus lactis and
Streptococcus thermophiles.
8. The method according to claim 1, wherein the comprises
Bifidobacterium breve and Bifidobacterium longum.
9. The method according to claim 1, wherein the composition
comprises 10-50 .mu.g beta-lactoglobulin-derived peptides per gram
total protein.
10. The method according to claim 1, wherein the allergy is cow's
milk protein allergy.
11. The method according to claim 1, wherein the Bifidobacterium
breve is the sole probiotic.
12. The method according to claim 3, wherein the composition
comprises 0.5-2 wt % of the prebiotic.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the National Phase of International Patent
Application No. PCT/NL2016/050190, filed Mar. 17, 2016, published
on Sep. 22, 2016 as WO 2016/148572 A1, which claims priority to
International Patent Application No. PCT/NL2015/050173, filed Mar.
18, 2015. The contents of these applications are herein
incorporated by reference in their entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted in ASCII format via EFS-WEB and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Sep. 12,
2017, is named 069818-3040Sequencelisting.txt and is 4 KB.
The invention is in the field of immunology and more particularly
relates to compositions for use in inducing oral tolerance and/or
for use in treatment and/or prevention (including reducing the risk
of occurrence) of allergy, in particular cow's milk protein
allergy. The invention rests particularly in the field of infants
and children.
BACKGROUND
One of the most common food allergies, especially in infancy and
childhood, is cow's milk allergy (CMA). Dietary proteins are
presented to the immune system via the gastrointestinal tract and
the normal response would be to elicit a tolerogenic immune
response to the ingested nutrients. This response is called oral
immune tolerance or oral tolerance. The induction of oral immune
tolerance is especially relevant for infants, who after birth are
exposed for the first time to dietary proteins and have to adapt to
this. If oral immune tolerance in infants is not established, food
allergy will occur.
About 2 to 3% of infants are allergic to cow's milk protein. For
infants suffering from allergy to cow's milk protein, infant
formulae are on the market comprising extensively hydrolysed
proteins (extensive protein hydrolysate) or even merely free amino
acids as nitrogen source. In these formulae no allergenic proteins
or peptides are present. Thereby exposure to milk protein is
avoided thus preventing a clinically manifested allergic reaction.
This is called a secondary prevention of cows' milk allergy.
However, as soon as cow's milk proteins are reintroduced into the
diet, the infant may again suffer from clinically manifested
allergic reactions.
Hypoallergenic formulae are on the market, comprising a partial
protein hydrolysate (partially hydrolysed proteins), which have a
decreased allergenicity. These formulations have the advantage that
they increase the likelihood of obtaining an immunological
tolerogenic response to cow's milk protein or peptides, with the
advantage that later on the native protein can be introduced in the
diet with a reduced risk of allergic reactions. This is called
primary prevention of cow's milk protein allergy and these formulae
are typically used for infants at risk of developing allergy.
SUMMARY OF THE INVENTION
The inventors found that a composition comprising a probiotic and a
beta-lactoglobulin-derived peptide comprising an amino acid
sequence corresponding to at least 8 consecutive amino acids of the
beta-lactoglobulin protein represented by SEQ ID No. 1 was
surprisingly effective in inducing oral tolerance and/or for use
and in treatment and/or prevention (including reducing the risk of
occurrence) of allergy, suitably food allergy, in particular cow's
milk protein allergy (CMA). Suitably, the composition further
comprises a prebiotic.
The present invention thus concerns a method for inducing oral
tolerance and/or treatment, prevention and/or reducing the risk of
occurrence of allergy in a subject, comprising administering to the
subject a composition comprising a probiotic and a
beta-lactoglobulin-derived peptide comprising an amino acid
sequence corresponding to at least 8 consecutive amino acids of the
beta-lactoglobulin protein represented by SEQ ID No. 1. Suitably,
the composition further comprises a prebiotic. In one embodiment,
the method is for inducing oral tolerance. In one embodiment, the
method is for treatment and/or prevention of allergy, suitably food
allergy, more suitably CMA.
The invention may also be worded as the use of a probiotic and a
beta-lactoglobulin-derived peptide comprising an amino acid
sequence corresponding to at least 8 consecutive amino acids of the
beta-lactoglobulin protein represented by SEQ ID No. 1 for the
manufacture of a composition for inducing oral tolerance and/or
treatment, prevention and/or reducing the risk of occurrence of
allergy in a subject. Suitably, the composition further comprises a
prebiotic. In one embodiment, the use is for the manufacture of a
composition for inducing oral tolerance. In one embodiment, the use
is for the manufacture of a composition for treatment and/or
prevention of allergy, suitably food allergy, more suitably
CMA.
In other words, the invention concerns a composition for use in
inducing oral tolerance and/or treatment, prevention and/or
reducing the risk of occurrence of allergy in a subject, said
composition comprising a probiotic and a beta-lactoglobulin-derived
peptide comprising an amino acid sequence corresponding to at least
8 consecutive amino acids of the beta-lactoglobulin protein
represented by SEQ ID No. 1. The composition suitably further
comprises a prebiotic. Worded differently, the invention pertains
to a composition comprising a probiotic and a
beta-lactoglobulin-derived peptide for use in oral tolerance,
and/or treatment, prevention or reducing the risk of allergy in a
subject, said peptide comprising an amino acid sequence
corresponding to at least 8 consecutive amino acids of the
beta-lactoglobulin protein represented by SEQ ID No. 1. Suitably,
the composition further comprises a prebiotic. In one embodiment,
the use is for inducing oral tolerance. In one embodiment, the use
is for treatment and/or prevention of allergy, suitably food
allergy, more suitably CMA.
The invention also concerns a composition comprising a probiotic
and a beta-lactoglobulin-derived peptide comprising an amino acid
sequence corresponding to at least 8 consecutive amino acids of the
beta-lactoglobulin protein represented by SEQ ID No. 1.
The invention also concerns a kit-of-parts comprising a first
container comprising infant nutrition and a second container
comprising a beta-lactoglobulin-derived peptide comprising an amino
acid sequence corresponding to at least 8 consecutive amino acids
of the beta-lactoglobulin protein represented by SEQ ID No. 1,
wherein the infant nutrition comprises a probiotic or wherein the
kit-of-parts comprises a third container comprising a
probiotic.
In one embodiment of the method, use, composition for use or
composition according to the invention, the
beta-lactoglobulin-derived peptide comprises an amino acid sequence
consisting of 12-30 consecutive amino acids from the region
spanning from amino acids 13 to 48 of the beta-lactoglobulin
protein represented by SEQ ID No. 1. In one embodiment of the
method, use, composition for use or composition according to the
invention, the beta-lactoglobulin-derived peptide consists of an
amino acid sequence selected from the group consisting of SEQ ID
No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, optionally
coupled to 1-6 further amino acids at its C- and/or N-terminus. In
one embodiment of the method, use, composition for use or
composition according to the invention, the composition further
comprises a prebiotic. In one embodiment of the method, use,
composition for use or composition according to the invention, the
prebiotic is selected from the group consisting of
fructo-oligosaccharide, non-digestible dextrin,
galacto-oligosaccharide, xylo-oligosaccharide,
arabino-oligosaccharide, arabino-galacto-oligosaccharide,
gluco-oligosaccharide, glucomanno-oligosaccharide,
galactomanno-oligosaccharide, mannan-oligosaccharide,
chito-oligosaccharide, uronic acid oligosaccharide,
sialyl-oligosaccharide and fuco-oligosaccharide. In one embodiment
of the method, use, composition for use or composition according to
the invention, the prebiotic comprises a mixture of a short-chain
oligosaccharide having an average degree of polymerisation of 2-8
and a long-chain oligosaccharide having an average degree of
polymerisation of 10-60. In one embodiment of the method, use,
composition for use or composition according to the invention, the
prebiotic comprises a galacto-oligosaccharide and/or a
fructo-oligosaccharide. In one embodiment of the method, use,
composition for use or composition according to the invention, the
probiotic comprises a strain of the genus Bifidobacteria,
Lactobacillus, or Streptococcus. In one embodiment of the method,
use, composition for use or composition according to the invention,
the probiotic comprises a strain selected from the group consisting
of Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium
infantis, Bifidobacterium bifidum, Lactobacillus acidophilus,
Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus
plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus,
Lactobacillus casei, Lactobacillus lactis and Streptococcus
thermophiles. In one embodiment of the method, use, composition for
use or composition according to the invention, the probiotic
comprises Bifidobacterium breve and/or Bifidobacterium longum. In
one embodiment of the method, use, composition for use or
composition according to the invention, the composition comprises
10-5000 .mu.g beta-lactoglobulin-derived peptides per gram total
protein. In one embodiment of the method, use or composition for
use according to the invention, the allergy is cow's milk protein
allergy.
DETAILED DESCRIPTION
The present invention concerns a method for inducing oral tolerance
and/or treatment and/or prevention of allergy in a subject, wherein
the method involves administration of a composition to said
subject, said composition comprising a probiotic and a
beta-lactoglobulin-derived peptide comprising an amino acid
sequence corresponding to at least 8 consecutive amino acids of the
beta-lactoglobulin protein represented by SEQ ID No. 1.
In the experiments, mice had been sensitized to intact whey protein
and consequently showed an acute allergic skin response after an
intradermal challenge with intact whey protein, i.e. they had
become whey protein allergic, which was also proven by an increase
in whey-specific IgE (data not shown). The inventors surprisingly
found that the allergic reaction to whey protein upon further
administration of whey protein was significantly suppressed (or
reduced) when the composition of the invention was administrated
prior to the whey protein challenge. In other words, administration
of the composition according to the invention induced oral
tolerance.
In this document and in its claims, the verb "to comprise" and its
conjugations is used in its non-limiting sense to mean that items
following the word are included, but items not specifically
mentioned are not excluded. In addition, reference to an element by
the indefinite article "a" or "an" does not exclude the possibility
that more than one of the element is present, unless the context
clearly requires that there be one and only one of the elements.
The indefinite article "a" or "an" thus usually means "at least
one".
In the context of the present invention, reference is made to the
following amino acid sequences:
TABLE-US-00001 SEQ ID Sequence No. 1 L I V T Q T M K G L D I Q K V
A G T W Y S L A M A A S D I S L L D A Q S A P L R V Y V E E L K P T
P E G D L E I L L Q K W E N G E C A Q K K I I A E K T K I P A V F K
I D A L N E N K V L V L D T D Y K K Y L L F C M E N S A E P E Q S L
A C Q C L V R T P E V D D E A L E K F D K A L K A L P M H I R L S F
N P T Q L E E Q C H I No. 2 Q K V A G T W Y S L A M A A S D I S No.
3 W Y S L A M A A S D I S L L D A Q S No. 4 A A S D I S L L D A Q S
A P L R V Y No. 5 L L D A Q S A P L R V Y V E E L K P
Composition
In a first aspect, the invention concerns a composition comprising
a probiotic and a beta-lactoglobulin-derived peptide comprising an
amino acid sequence corresponding to at least 8 consecutive amino
acids of the beta-lactoglobulin protein represented by SEQ ID No.
1. The composition according to the invention is to be used in the
method or use or composition for use according to the invention,
which involve administration of the composition according to the
invention. The composition according to the invention may be used
as a pharmaceutical product or a nutritional product.
The probiotic and the beta-lactoglobulin-derived peptide, and
suitably the prebiotic, are present in therapeutically effective
amounts.
In one aspect, the composition according to the invention may be
used as a pharmaceutical product comprising one or more
pharmaceutically acceptable carrier materials. Such product may
contain the daily dosages as defined below in one or more dosage
units. The dosage unit may be in a liquid form or in a solid form,
wherein in the latter case the daily dosage may be provided by one
or more solid dosage units, e.g. in one or more capsules or
tablets. The pharmaceutical product, suitably for enteral
application, may be a solid or liquid galenical formulation.
Examples of solid galenical formulations are tablets, capsules
(e.g. hard or soft shell gelatine capsules), pills, sachets,
powders, granules and the like which contain the active ingredients
together with conventional galenical carriers. Any conventional
carrier material can be utilized. The carrier material can be
organic or inorganic inert carrier material suitable for oral
administration. Suitable carriers include water, gelatine, gum
arabic, lactose, starch, magnesium stearate, talc, vegetable oils,
and the like. Additionally, additives such as flavouring agents,
preservatives, stabilizers, emulsifying agents, buffers and the
like may be added in accordance with accepted practices of
pharmaceutical compounding. While the individual active ingredients
are suitably administered in a single composition, they may also be
administered in individual dosage units.
In one aspect, the composition according to the invention may be
used as a nutritional product, for example as a nutritional
supplement, e.g. as an additive to a normal diet, as a fortifier,
to add to a normal diet, as a complete nutrition or as infant
nutrition suitable for feeding infants (e.g. infant formula or
follow-on formula). The nutritional product suitably comprises
fats, proteins, and carbohydrates. It is understood that a
nutritional product differs from a pharmaceutical product by the
presence of nutrients which provide nutrition to the subject to
which the composition is administered, for instance the presence of
protein, fat, digestible carbohydrates and dietary fibres. It may
further contain ingredients such as minerals, vitamins, organic
acids, and flavouring agents. Although the term "nutraceutical
product" is often used in literature, it denotes a nutritional
product with a pharmaceutical component or pharmaceutical purpose.
Hence, the nutritional composition according to the invention may
also be used in a nutraceutical product.
The composition of the invention is typically an enteral
composition, i.e. intended for oral administration. It is suitably
administered in liquid form. For instance, the composition may
comprise water in which the further components are dissolved or
suspended. The composition is thus suitably a liquid, or a solid
(typically a powder or tablet) which is reconstitutable with a
liquid, suitably with water, to obtain a liquid composition.
Suitably the liquid composition has a viscosity below 100 mPas,
more suitably below 60 mPas, more suitably below 35 mPas, even more
suitably below 6 mPas as measured in a Brookfield viscometer at
20.degree. C. at a shear rate of 100 s.sup.-1.
The composition typically comprises a lipid fraction, a protein
component and a digestible carbohydrate component. The caloric
content of the composition, when in liquid form, suitably comprises
60 to 85, more suitably 60 to 70 kcal/100 ml liquid. The osmolarity
of the present composition is suitably between 150 and 420
mOsmol/l, more suitably 260 to 360 mOsmol/l.
Suitably the lipid component provides 2.9 to 6 g lipid per 100
kcal, suitably the protein component provides 1.8 to 5.5 g per 100
kcal, suitably 1.8 to 2.5 g per 100 kcal and suitably the
digestible carbohydrate component provides 9 to 14 g per 100 kcal,
of the composition. The amount of total calories is determined by
the sum of calories derived from protein, lipids, digestible
carbohydrates and non digestible oligosaccharides.
Protein
The composition according to the invention comprises a protein
fraction, which at least includes the aforementioned
beta-lactoglobulin-derived peptide. Beta-lactoglobulin is one of
two major whey proteins in the milk of cows and sheep but is not
found in human milks. Often in case of cow's milk protein allergy
beta-lactoglobulin is the allergen.
The beta-lactoglobulin-derived peptide according to the invention
is a peptide comprising an amino acid sequence corresponding to at
least 8, suitably 10-50 amino acids, more suitably 12-30, more
suitably 14-25, more suitably 16-20, most suitably 18 consecutive
amino acids of the beta-lactoglobulin protein represented by SEQ ID
No. 1.
In the context of the invention, an amino acid sequence
"corresponding to" the beta-lactoglobulin protein allows for at
most three, more suitably at most two, even more suitably one amino
acid substitution in the amino acid sequence may be allowed for,
said substitution(s) suitably being conservative amino acid
substitution(s). A "conservative amino acid substitution" refers to
the interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulphur-containing
side chains is cysteine and methionine. Suitable conservative amino
acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine. Substitutional variants of the amino acid
sequence disclosed herein are those in which at least one residue
in the disclosed sequences has been removed and a different residue
inserted in its place. Suitably, the amino acid change is
conservative. Suitable conservative substitutions for each of the
naturally occurring amino acids are as follows: Ala to Ser; Arg to
Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn;
Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu
to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to
Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or
Phe; and, Val to Ile or Leu. In particular, the Glu (E) to Gln (Q)
substitution of amino acid 45 within SEQ ID No. 1 is covered by the
present invention. SEQ ID No. 1 represents the B variant of the
beta-lactoglobulin protein, while Glu to Gln substitution at amino
acid 45 gives the D variant, which are both suitably used in the
context of the present invention. In one embodiment, no
modifications including conservative amino acid substitutions other
than the Glu to Gln substitution of amino acid 45 within the
suitable amino acid sequence regions of SEQ ID No. 1 is allowed
for. In one embodiment, no modifications including conservative
amino acid substitutions in the suitable amino acid sequence
regions is allowed for.
Suitably, the amino acid sequence contains consecutive amino acids
from the region spanning from amino acids 3 to 117, more suitably
amino acids 10 to 63, most suitably amino acids 13 to 48, of the
beta-lactoglobulin protein.
Suitably, the peptide has a molecular weight of at most 5 kDa, in
particular from 0.1 to 4.9 kDa, suitably from 0.5 to 4.5, more
suitably of 2 to 4 kDa, most suitably of about 2.4 kDa. In a
suitable embodiment the beta-lactoglobulin-derived peptide consists
of 12 to 30 amino acids, suitably 14 to 25 amino acids, more
suitably 16 to 20 amino acids, most suitably 18 amino acids.
Suitable beta-lactoglobulin-derived peptides comprise, most
suitably consist of, the amino acid sequences represented by SEQ ID
No. 2 (amino acids 13 to 30 of SEQ ID No. 1), SEQ ID No. 3 (amino
acids 19 to 36 of SEQ ID No. 1), SEQ ID No. 4 (amino acids 25 to 42
of SEQ ID No. 1) and SEQ ID No. 5 (amino acids 31 to 48 of SEQ ID
No. 1), wherein amino acid 15 in SEQ ID No. 5 (corresponding to
amino acid 45 in SEQ ID No. 1) may be substituted with Gln (Q).
The beta-lactoglobulin-derived peptide may comprise further amino
acids at the C- and/or N-terminus of the amino acid sequence
corresponding to at least 8 consecutive amino acids of the
beta-lactoglobulin protein. In other words, the amino acid sequence
corresponding to at least 8 consecutive amino acids of the
beta-lactoglobulin protein is optionally coupled to further amino
acids at its C- and/or N-terminus. Typically, 1-6 further amino
acids may be present, suitably 1-5, more suitably 1-4, most
suitably 1-3 further amino acids may be present, which can be any
(combination of) amino acid(s). In one embodiment, no further amino
acids are present at the C- and/or N-terminus of the at least 8
consecutive amino acids of the beta-lactoglobulin protein.
Suitably, the beta-lactoglobulin-derived peptide consists of an
amino acid sequence selected from the group consisting of SEQ ID
No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 5
wherein amino acid 15 is substituted with Gln (Q). Most suitably,
the beta-lactoglobulin-derived peptide consists of an amino acid
sequence selected from the group consisting of SEQ ID No. 2, SEQ ID
No. 3, SEQ ID No. 4 and SEQ ID No. 5.
The composition may comprise more than one distinct
beta-lactoglobulin-derived peptide, such as 2, 3, 4, 5 or even
more, e.g. up to 2 or up to 10, distinct beta-lactoglobulin-derived
peptides. Suitably, the composition comprises 1-10 distinct
beta-lactoglobulin-derived peptides, more suitably 2-5 distinct
beta-lactoglobulin-derived peptides, most suitably 4 distinct
beta-lactoglobulin-derived peptides. Herein, each distinct
beta-lactoglobulin-derived peptide comprises a different amino acid
sequence of the beta-lactoglobulin protein represented by SEQ ID
No. 1. The difference may reside in the number of amino acids from
the beta-lactoglobulin protein sequence and/or in the location of
the amino acid sequence within the beta-lactoglobulin protein
sequence, suitably it resides in the location of the amino acid
sequence within the beta-lactoglobulin protein.
The mixture of more than one distinct beta-lactoglobulin-derived
peptide suitably comprises at least one, more suitably 2-4, even
more suitably 3 or 4, most suitably all 4,
beta-lactoglobulin-derived peptide(s) consisting of an amino acid
sequence selected from the group consisting of SEQ ID No. 2, SEQ ID
No. 3, SEQ ID No. 4 and SEQ ID No. 5.
In accordance with the present invention, the
beta-lactoglobulin-derived peptides can be chemically synthesised
as known in the art, or isolated after expression by a genetically
modified host such as an E. Coli strain or Lactobacillus strain.
Alternatively the peptides are isolated and purified from a whey
protein or beta-lactoglobulin hydrolysate.
The composition according to the invention may comprise further
proteinaceous material, in addition to the
beta-lactoglobulin-derived peptide(s), although the
beta-lactoglobulin-derived peptide(s) may also be the only protein
source. In the context of the present invention the additional
"protein" or "proteinaceous material" encompasses proteins,
peptides, free amino acids and partially or extensively hydrolysed
proteins. Typically, extensive protein hydrolysates have a free
amino acid content of above 10 g per 100 g protein. Any additional
extensively hydrolysed protein in the present invention suitably
relates to protein which has been hydrolysed and has less than 3 wt
% of peptides with a size above 5 kDa. Typically, extensively
hydrolysed protein has been obtained by protease hydrolysis
followed by an ultrafiltration step by filtrating over a membrane
with a cut-off of 2-5 kDa. Suitably these extensive protein
hydrolysates comprise almost no peptides with a size over 1.5
kDa.
Suitably, the further proteinaceous material--additional to the
beta-lactoglobulin-derived peptide--does not evoke an allergic
reaction, such as free amino acids, partially hydrolysed protein
and/or extensively hydrolysed protein. As a further protein
component, i.e. apart from the beta-lactoglobulin-derived peptide,
the composition according to the present invention suitably
comprises free amino acids, partially hydrolysed whey protein
and/or extensively hydrolyzed whey proteins.
In some embodiments, the composition according to the present
invention does not contain intact cow's milk protein. The
composition may comprise an additional protein component selected
from the group consisting of free amino acids, extensively
hydrolysed whey protein and proteins from other sources such as
soy, pea, rice, collagen or the like, in intact form, in partially
hydrolysed form, and/or in extensively hydrolysed form.
The present composition suitably contains at least 50 wt % protein
component derived from non-human milk, more suitably at least 90 wt
%, based on dry weight of total protein.
The present composition suitably contains 4 to 25%, more suitably 5
to 20%, more suitably 7 to 16%, most suitably 7 to 12% protein,
based on total calories. The present composition, when in liquid
form, suitably contains 0.5 to 6.0 g, more suitably 0.8 to 3.0 g,
even more suitably 1.0 to 2.5 g of protein per 100 ml. The present
composition suitably comprises at least 7.0 wt %, more suitably at
least 8.0 wt %, most suitably at least 9 or at least 10 wt %
protein based on dry weight of the total composition. Suitably, the
present composition comprises at most 40 wt %, more suitably at
most 15 wt %, suitably at most 20 wt % of protein based on dry
weight of the total composition.
In a suitable embodiment of the present invention, the present
composition comprises at least 10 .mu.g, more suitably at least 30
.mu.g, suitably at least 50 .mu.g, suitably 10 to 5000 .mu.g, more
suitably 20 to 2000 .mu.g, more suitable 30 to 500 .mu.g and
particularly suitably 50 to 250 .mu.g of the
beta-lactoglobulin-derived peptide per gram of total protein.
Carbohydrate
The composition according to the invention may comprise a
carbohydrate fraction, which may include a prebiotic. In the
context of the present invention, the term "prebiotic" refers to
one or more non-digestible oligosaccharides. Advantageously, the
non-digestible oligosaccharide is water-soluble (according to the
method disclosed in L. Prosky et al, J. Assoc. Anal. Chem 71:
1017-1023, 1988). Non-digestible oligosaccharides are not digested
in the intestine by the action of digestive enzymes present in the
human upper digestive tract (small intestine and stomach) but
instead are fermented by the human intestinal microbiota.
Suitable non-digestible oligosaccharides are selected from the
group consisting of fructo-oligosaccharide, non-digestible dextrin,
galacto-oligosaccharide, xylo-oligosaccharide,
arabino-oligosaccharide, arabinogalactooligosaccharide,
gluco-oligosaccharide, glucomannooligosaccharide,
galactomanno-oligosaccharide, mannanoligosaccharide,
chito-oligosaccharide, uronic acid oligosaccharide,
sialyl-oligosaccharide and fuco-oligosaccharide. Especially
suitable non-digestible oligosacchardies are
fructo-oligosaccharides and/or galacto-oligosaccharides. The
oligosaccharides suitably have a degree of polymerization of 2-200.
In one embodiment, fructo-oligosaccharides and
fructo-polysaccharides (and mixtures thereof) with a DP of 2-200
are suitable prebiotics in the context of the invention.
One suitable type of oligosaccharide is a short-chain
oligosaccharide which has an average degree of polymerisation of
less than 10, suitably at most 8, suitably in the range of 2-7. The
short-chain oligosaccharide suitably comprises
galacto-oligosaccharides and/or fructo-oligosaccharides. In one
embodiment, the composition comprises galacto-oligosaccharides, in
particular .beta.-galacto-oligosaccharides, more in particular
trans-galacto-oligosaccharides. The galacto-oligosaccharides
suitably have an average degree of polymerisation in the range of
2-8, i.e. are short-chain oligosaccharides in the context of the
invention.
Suitably, the composition comprises short-chain
fructo-oligosaccharides and/or short-chain
galacto-oligosaccharides, suitably at least short-chain
fructo-oligosaccharides. (trans)galactooligosaccharide is for
example available under the trade name Vivinal.RTM. GOS (Borculo
Domo Ingredients, Zwolle, Netherlands), Bimuno (Clasado), Cup-oligo
(Nissin Sugar) and Oligomate55 (Yakult). Fructooligosaccharides may
be inulin hydrolysate products having an average DP within the
aforementioned (sub-) ranges; such FOS products are for instance
commercially available as Raftilose P95 (Orafti) or with
Cosucra.
Another suitable type of oligosaccharide is long-chain
fructo-oligosaccharides which has an average degree of
polymerisation above 10, typically in the range of 10-100, suitably
15-50, most suitably above 20. A particular type of long-chain
fructo-oligosaccharides is inulin, such as Raftilin HP.
The present composition may contain one type of non-digestible
oligosaccharide or a mixture of two or more types of non-digestible
oligosaccharides, suitably it comprises a mixture of two or more
non-digestible oligosaccharides, most suitably a mixture of two
non-digestible oligosaccharides. In case the prebiotic contains or
consists of a mixture of two distinct oligosaccharides, one
oligosaccharide may be short-chain as defined above and one
oligosaccharide may be long-chain as defined above. Suitably,
short-chain oligosaccharides and long-chain oligosaccharides are
present in a weight ratio short-chain to long-chain in the range of
1:99-99:1, more suitably 1:1-99:1, more suitably 4:1-97:3, even
more suitably 5:1-95:5, even more suitably 7:1-95:5, even more
suitably 8:1-10:1, most suitably about 9:1.
In one embodiment, the prebiotic comprises a mixture of
fructo-oligosaccharides and/or galacto-oligosaccharides. Suitable
mixtures include mixtures of long-chain fructo-oligosaccharides
with short-chain fructo-oligosaccharides or short-chain
galacto-oligosaccharides, most suitably long-chain
fructo-oligosaccharides with short-chain
fructo-oligosaccharides.
In one embodiment, the prebiotic comprises a mixture of
fructo-oligosaccharides, most suitably a mixture of short-chain
fructo-oligosaccharides (sc-FOS) and long-chain
fructo-oligosaccharides (lc-FOS). These fructo-oligosaccharides
suitably account for at least 80 wt %, more suitably at least 90 wt
% of the prebiotic. In a most suitable embodiment, the prebotic
fraction consists of a mixture of sc- and lc-FOS.
The prebiotics may be present in the composition at any suitable
concentration, suitably. The present composition suitably comprises
0.05 to 20 wt % of said non-digestible oligosaccharides, more
suitably 0.5 to 15 wt %, even more suitably 1 to 10 wt %, most
suitably 2 to 10 wt %, based on dry weight of the present
composition. When in liquid form, the present composition suitably
comprises 0.01 to 2.5 wt % non-digestible oligosaccharide, more
suitably 0.05 to 1.5 wt %, even more suitably 0.25 to 1.5 wt %,
based on 100 ml.
The composition according to the invention may comprise further
carbohydrates, suitably the present composition comprises a
digestible carbohydrate. Typically, digestible carbohydrates that
are known in the art to be suitable for use in infant nutritional
compositions are used. Suitably, the digestible carbohydrate is
selected from digestible polysaccharides (e.g. starch,
matodextrin), digestible monosaccharides (e.g. glucose, fructose),
and digestible disaccharides (e.g. lactose, sucrose). Particularly
suitable is lactose and/or maltodextrin. In one embodiment, the
composition does not comprise lactose.
The digestible carbohydrate component suitably comprises at least
60 wt % lactose based on total digestible carbohydrate, more
suitably at least 75 wt %, even more suitably at least 90 wt %
lactose based on total digestible carbohydrate
Lipid
The composition according to the invention suitably comprises a
lipid component, suitably a lipid component suitable for infant
nutrition as known in the art. The lipid component of the present
composition suitably provides 2.9 to 6.0 g, more suitably 4 to 6 g
per 100 kcal of the composition. When in liquid form, the
composition suitably comprises 2.1 to 6.5 g lipid per 100 ml, more
suitably 3.0 to 4.0 g per 100 ml. Based on dry weight the present
infant or follow on formula suitably comprises 12.5 to 40 wt %
lipid, more suitably 19 to 30 wt %.
The lipid component typically comprises the essential fatty acids
alpha-linolenic acid (ALA), linoleic acid (LA) and suitably long
chain polyunsaturated fatty acids (LC-PUFA). The LC-PUFA, LA and/or
ALA may be provided as free fatty acids, in triglyceride form, in
diglyceride form, in monoglyceride form, in phospholipid form, or
as a mixture of one of more of the above. Suitably the present
composition contains at least one, suitably at least two lipid
sources selected from the group consisting of rape seed oil (such
as colza oil, low erucic acid rape seed oil and canola oil), high
oleic sunflower oil, high oleic safflower oil, olive oil, marine
oils, microbial oils, coconut oil, palm kernel oil and milk
fat.
Probiotic
The composition according to the invention comprises a probiotic.
In the context of the present invention, the term "probiotic"
refers to a strain of probiotic bacteria. Probiotic bacteria are
known in the art. Suitably, the probiotic bacteria are not
genetically modified.
Suitable probiotic bacteria include bacteria of the genus
Bifidobacteria (e.g. B. breve, B. longum, B. infantis, B. bifidum),
Lactobacillus (e.g. L. Acidophilus, L. paracasei, L. johnsonii, L.
plantarum, L. reuteri, L. rhamnosus, L. casei, L. lactis), and
Streptococcus (e.g. S. thermophilus). B. breve and B. longum are
especially suitable probiotics.
Most suitably, the probiotic comprises a strain of B. breve. The B.
breve suitably has at least 95% identity of the 16 S rRNA sequence
when compared to the type strain of B. breve ATCC 15700, more
suitably at least 97% identity (Stackebrandt & Goebel, 1994,
Int. J. Syst. Bacteriol. 44:846-849). Suitable B. breve strains may
be isolated from the faeces of healthy human milk-fed infants.
Typically, these are commercially available from producers of
lactic acid bacteria, but they can also be directly isolated from
faeces, identified, characterised and produced. According to one
embodiment, the present composition contains a B. breve selected
from the group consisting of B. breve Bb-03 (Rhodia/Danisco), B.
breve M-16V (Morinaga), B. breve R0070 (Institute Rosell,
Lallemand), B. breve BR03 (Probiotical), B. breve BR92) (Cell
Biotech), DSM 20091, LMG 11613, YIT4065, FERM BP-6223 and CNCM
I-2219. Most suitably, the B. breve is selected from the group
consisting of B. breve M-16V and B. breve CNCM I-2219, most
suitably B. breve M-16V. B. breve I-2219 was published in WO
2004/093899 and was deposited at the Collection Nationale de
Cultures de Microorganisms, Institute Pasteur, Paris, France on 31
May 1999 by Compagnie Gervais Danone. B. breve M-16V was deposited
as BCCM/LMG23729 and is commercially available from Morinaga Milk
Industry Co., Ltd.
The combination of a prebiotic and a probiotic is also referred to
as a "synbiotic".
The probiotic may be present in the composition at any suitable
concentration, suitably in a therapeutically effective amount or
"amount effective for treating" in the context of the invention.
Suitably, the probiotic is included in the present composition in
an amount of 10.sup.2-10.sup.13 cfu per g dry weight of the
composition, suitably 10.sup.5-10.sup.12 cfu/g, most suitably
10.sup.7-10.sup.10 cfu/g.
Application
The composition according to the invention is for inducing oral
tolerance and/or treating, preventing and/or reducing the risk
occurrence of allergy in a subject. The allergy may be cow's milk
protein allergy, suitably allergy to whey protein. (Prophylactic)
treatment of allergy preferably involves reducing the (acute)
symptoms associated with ingesting an allergen, in particular
wherein the allergen is cow's milk protein. Suitably, the (acute)
symptoms are reduced when the allergen is ingested again. The
allergen is suitably cow's milk protein. In the context of the
present invention the term "treatment" is understood to mean a
therapeutic treatment of a human or animal patient, suitably
humans, in particular infants, in terms of partially or completely
curing the allergy and/or to alleviate or ameliorate symptoms of
the allergy. Suitably, the treatment is an oral immuno-therapy. In
the context of the invention, "prevention" may also be referred to
as "reducing the risk or occurrence of", and is understood to mean
a prophylactic treatment of a human or animal patient, suitably a
human, in particular an infant.
The composition according to the invention can be used as a
nutritional composition, nutritional therapy, nutritional support,
as a medical food, as a food for special medical purposes or as a
nutritional supplement. The present composition is suitably an
enteral composition. The composition is administered to, or
intended to be administered to, a subject in need thereof, in
particular to children and infants, including toddlers, suitably
children up to 6 years of age, suitably infants typically with an
age of 0-36 month, more suitably 0-12 months of age, most suitably
0-6 months of age. Thus, in some embodiments, the present
composition is an infant formula, follow-on formula or growing-up
milk, most suitably it is an infant formula.
In a particular embodiment, the composition is for administration
to subjects, in particular infants, at risk of developing allergy
or suffering from allergy, especially cow's milk protein allergy.
Infants that are known to be at risk of developing allergy include
infants born from at least one parent suffers from, or has suffered
from, atopic disorders (e.g. eczema) and/or allergy, most in
particular from CMA.
The present composition is suitably administered in a daily dose of
0.01 mg-1 g beta-lactoglobulin-derived peptides, more suitably
0.1-100 mg, even more suitably 0.5-5 mg, most suitably 1-2.5
mg.
In a further aspect, the present invention further relates to a
kit-of-parts comprising or consisting of the following two or three
different containers and instructions for use: A first container
comprising infant nutrition, a second container comprising a
beta-lactoglobulin-derived peptide as defined hereinabove and
optionally a third container comprising a probiotic as defined
hereinabove. Alternatively, the probiotic can be comprised in the
first container.
The infant nutrition is suitably an infant formula, follow-on
formula or growing-up milk as known in the art. Most suitably, the
infant nutrition is specifically targeted for allergic infants
and/or infants at risk of developing allergy, in particular wherein
the allergy is CMA. Such allergic formulae are known in the art.
The infant nutrition may also be referred to as the composition
according to the invention as defined hereinabove, albeit without
comprising a beta-lactoglobulin-derived peptide and optionally
without comprising a probiotic.
The infant nutrition may or may not comprise the probiotic as
defined above. In case the infant nutrition does not contain the
probiotic, the kit of parts comprises a third container comprising
the probiotic. The third container is typically in the form of a
sachet or stickpack and suitably comprises a powder consisting of
the probiotic and a acceptable carrier, typically lactose. In case
the infant nutrition contains the probiotic, the kit may be limited
to the first and second container. The second container is
typically in sachet or stickpack and suitably comprises a powder
consisting of a beta-lactoglobulin-derived peptide and a acceptable
carrier, typically lactose. The instructions for use conveniently
instruct the user to combine the contents of the two or three
containers in the appropriate format and reconstitute the resulting
mixture with a liquid, typically water, to obtain a ready-to-use
liquid composition.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the skin response (ear swelling) results of example 1.
The tested groups are: 1=negative control; 2=positive control;
3=peptide-diet group; 4=synbiotics-diet group;
5=peptide+synbiotics-diet group.
FIG. 2 shows the anaphylactic shock scores of example 1. The tested
groups are: 1=negative control; 2=positive control; 3=peptide-diet
group; 4=synbiotics-diet group; 5=peptide+synbiotics-diet
group.
FIG. 3 shows the T cell subset analysis results of example 3.
Lymphocytes isolated from the small intestine lamina propria
(SI-LP) were analysed by flow cytometry for T.sub.h1 and T.sub.h2
phenotypes. The tested groups are: 1=negative control; 2=positive
control; 3=peptide-diet group; 4=synbiotics-diet group;
5=peptide+synbiotics-diet group. Test groups fed a synbiotics diet
are shown with a solid grey bar. For each tested group the
percentage of CD4.sup.+ cells with an activated T.sub.h1 phenotype
(Graph A) or with an activated T.sub.h2 phenotype (Graph B) are
shown, as well as the T.sub.h1/T.sub.h2 ratios within an individual
CD4.sup.+ population (Graph C). Data are presented as the
mean.+-.SEM of n=4 in the negative control group and n=6-8 in all
other groups. * p<0.05, ** p<0.01 as analysed with ANOVA
followed by Bonferroni post hoc test for selected groups.
FIG. 4 shows the ex vivo splenocyte cytokine production results of
example 3. The tested groups are: 1=negative control; 2=positive
control; 3=peptide-diet group; 4=synbiotics-diet group;
5=peptide+synbiotics-diet group. Test groups fed a synbiotics diet
are shown with a solid grey bar. The tested cytokines are: IL-13
(Graph A), IL-10 (Graph B), IL-5 (Graph C), IL-17A (Graph D) and
IFN-.gamma. (Graph E). Data are presented as the mean.+-.SEM of n=4
in the PBS/CT group and n=6-8 in all other groups. * p<0.05, **
p<0.01 as analysed with Kruskal-Wallis non-parametric test,
followed by Dunn's post hoc test for selected groups.
FIG. 5 shows the skin response (ear swelling) results of example 4.
The tested groups are: 1=negative control; 2=positive control;
3=peptide-diet group (solid grey bar); 4=probiotics-diet group
(diagnonally striped bar); 5=peptide+probiotics-diet group
(diagnonally striped bar and a grey background). Data are presented
as the mean.+-.SEM of n=6-8 in all other groups. **** p<0.0001,
** p<0.01 as analysed with ANOVA followed by Dunnette's post hoc
test for selected groups.
EXAMPLES
Example 1
Material and Methods
Peptides:
18-amino acid (AA)-long peptides from beta-lactoglobulin were
synthetically produced by JPT Peptide Technologies (Berlin,
Germany). The synthetic peptides contained a 12-AA-long overlap and
their sequence was spanning the B variant of beta-lactoglobulin.
The four peptides were previously screened in an assay with human T
cell lines by Meulenbroek et al. (Pediatr. Allergy Immunol.
2013:7:656-664) and were selected based on their T cell reactivity
for further testing in animal models. Prior to the animal
experiment peptides were dissolved in PBS and a mixture of all four
peptides was prepared in which each peptide was at a concentration
of 0.08 mg/ml ("PepMix"). Peptide sequences are given in Table
1.
TABLE-US-00002 TABLE 1 Peptide sequences Peptide Sequence 1 (18 AA)
SEQ ID No. 2 QKVAGTWYSLANIAASDIS 2 (18 AA) SEQ ID No. 3
WYSLANIAASDISLLDAQS 3 (18 AA) SEQ ID No. 4 AASDISLLDAQSAPLRVY 4 (18
AA) SEQ ID No. 5 LLDAQSAPLRVYVEELKP
Diets:
Semipurified cow's milk protein-free standard mouse chow was
composed based on AIN-93G recipe (control diet) and supplemented
with synbiotics (synbiotic diet) (Research Diet Services, Wijk bij
Duurstede, The Netherlands). The synbiotic supplementation
consisted of 1 wt % of non-digestible short-chain (sc-) and
long-chain (lc-) fructo-oligosaccharides (FOS) (Raftilose P95
(Orafti) and Raftiline HP, respectively) in a ratio scFOS/lcFOS=9:1
and 2 wt % 2.times.10.sup.9 CFU/g Bifidobacterium breve M-16V
(Morinaga Milk Industry Co., Ltd, Tokyo, Japan). The synbiotic
components were mixed through the diet and the mixture was pressed
into pellets. Diets were stored at -4.degree. C. prior to use.
Animals:
Three-week-old pathogen-free female C3H/HeOuJ mice were purchased
from Charles River Laboratories (Sulzfeld, Germany) and were
maintained on a cow's milk protein-free standard mouse chow
(AIN-93G soy, Research Diet Services). Mice were housed in the
animal facility at Utrecht University. Animal care and use was in
accordance with the guidelines of the Dutch Committee of Animal
Experiments.
Study Protocol:
In order to investigate the tolerogenic properties of the synthetic
peptides in combination with the synbiotic diet, a murine model for
cow's milk allergy was used as described by Van Esch et al.
(Pediatr Allergy Immunol 2011; 22:820-826). Mice were orally
exposed (using a blunt needle) to 0.5 ml of the PepMix or phosphate
buffered saline (PBS, Lonza, Walkerville, Md., USA) prior to
sensitization (daily; from day -7 to day -2). In the same week
(from day -9 to day 0) mice were fed the control diet or the
synbiotic diet ad libitum. Subsequently, on day 0, 7, 14, 21 and
28, mice were sensitized orally with 20 mg whey protein (DMV
International, Veghel, The Netherlands) homogenized in 0.5 ml PBS
and mixed with 10 .mu.g cholera toxin (CT; List Biological
Laboratories, Inc. California, USA) as an adjuvant. The
non-sensitized mice received 10 .mu.g cholera toxin in 0.5 ml PBS
only. Table 2 summarizes the five groups tested.
Five days after the last sensitization, mice underwent an
intradermal whey challenge (injection in the ear pinnae with 10
.mu.g whey protein in 20 .mu.l PBS) and the acute allergic skin
response was recorded. The ear thickness was measured in duplicate
before and 1 h after the intradermal challenge using a digital
micrometer (Mitutoyo, Veenendaal, The Netherlands). The
allergen-specific ear swelling is the difference between the
average ear thickness at 1 h and the average basal ear thickness
(.DELTA.=ear thickness at 1 h-basal ear thickness) and is expressed
in micrometer. The ear swelling due to the local injection is
reflected in the ".DELTA. ear swelling" of the non-sensitized mice
(Group 1). Next to the ear swelling, clinical symptoms, such as
anaphylactic shock, were monitored and scored according to a table
as previously described Van Esch et al. (Pediatr Allergy Immunol
2011; 22:820-826).
TABLE-US-00003 TABLE 2 Interventions in the different groups Group
Pre-treatment Sensitization Challenge 1 negative control PBS +
control diet PBS + CT whey 2 positive control PBS + control diet
whey + CT whey 3 peptide PepMix + control diet whey + CT whey 4
synbiotics PBS + synbiotic diet whey + CT whey 5 peptide + PepMix +
synbiotic whey + CT whey synbiotics diet
Statistical Analysis:
All statistical analyses were conducted using GraphPad Prism 6.0c
software. Data was analysed with one-way ANOVA and post hoc
Bonferroni's multiple comparison test. The anaphylactic shock
scores were analysed using Kruskal-Wallis test because of the
non-parametric nature of the data. All data is presented as
mean.+-.SEM of 5-8 animals per group. P<0.05 was considered of
statistical significance.
Results
Skin response results are summarized in FIG. 1 and the results on
anaphylactic shock scores in FIG. 2. Sensitized, but untreated
animals (Group 2) developed a significantly higher allergic
response compared to the non-sensitised ones (Group 1). The
allergic response was significantly reduced after pre-exposing the
animals to a mixture of tolerogenic peptides combined with a diet
containing synbiotics (Group 5). The treatments with the peptide
mixture (Group 3) and with synbiotic diet (Group 4) were
insufficient to reduce the allergic response when applied alone and
only the combined exposure showed to be effective (Group 5).
Furthermore, sensitized animals developed significant anaphylactic
shock symptoms 20-40 min after the intradermal injection with the
allergen. However, only the combined peptides-synbiotics (Group 5)
exposure prevented from developing significantly higher
anaphylactic shock symptoms when compared to the non-sensitized
controls (Group 1). Anaphylactic shock scores were significantly
increased in Groups 2, 3 and 4, compared to Group 1.
The above experiment was repeated with the exception that the 1% wt
% sc-FOS/1c-FOS was substituted with 1 wt % of
sc-galacto-oligosaccharides (GOS) and lc-FOS (Vivinal.RTM. GOS and
Raftiline HP, respectively) in a ratio scGOS/lcFOS=9:1 for the
synbiotic that was fed to the animals. The results from this
experiment suggest that pre-exposure of animals with a combination
of this synbiotic and the tolerogenic peptide mixture also reduces
the allergic response to intradermal whey challenged, as compared
to a control.
Example 2
An infant formula for infants at risk of cow's milk protein allergy
or for infants allergic to cow's milk protein: Energy density:
0.6-0.77 kcal/ml. Protein is present in the form of free amino
acids and the beta-lactoglobulin-derived peptides of the present
invention. Per g protein about 100 .mu.g peptide mix as tested in
example 1 is present. Further characteristics are given in Table
3.
TABLE-US-00004 TABLE 3 Nutritional information per 100 ml* Energy
(kJ) 293 Energy (kcal) 70 protein (g) 1.9 (11 en %) carbohydrate
(g) 7.9 (45 en %) lipids (g) 3.4 (44 en %) LA (g) 0.6 ALA (mg) 60
AA (mg) 12 DHA (mg) 7 Pepmix of example 1 (.mu.g) 190 B. breve 2
.times. 10.sup.7 cfu *or per 14.7 g powder to be reconstituted in
water to a total of 100 ml
The composition further comprises minerals, vitamins as prescribed
for infant formulae, and has an osmolarity of 324 mOsm/L.
Example 3
Materials and Methods
Peptides, Diets, Animals and Treatment Protocol: Same as in Example
1.
Cell Isolation from Tissues:
Lymphocytes were isolated from spleen, mesenteric lymph nodes (MLN)
and small intestine lamina propria. Spleens and MLN were crushed
through 70 .mu.m cell strainers. Splenocyte suspension was
incubated for 5 min. on ice with lysis buffer to remove red blood
cells. Both splenocytes and MLN cells were taken up in RPMI 1640
supplemented with 10% FCS and penicillin (100 U/mL)/streptomycin
(100 .mu.g/mL). For the isolation of lamina propria cells, the
whole small intestine was removed, cleared of Peyer's patches (PP),
washed in cold PBS, opened longitudinally, and minced in 0.5 cm
fragments. Samples were then washed in Hank's Balanced Salt
Solution (HBSS; Invitrogen, Life Technologies, Carlsbad, Calif.,
USA) supplemented with 15 .mu.M HEPES (Gibco, Life Technologies,
Carlsbad, Calif., USA), pH=7.2 followed by 4.times.15 min
incubations with HBSS supplemented with 15 .mu.M HEPES, 5 .mu.M
Naz-EDTA, 10% FCS and penicillin (100 U/mL)/streptomycin (100
.mu.g/mL), pH=7.2. The fragments were then washed in RPMI 1640
supplemented with 5% FCS and penicillin (100 U/mL)/streptomycin
(100 .mu.g/mL) and incubated 2.times.45 min with an enzyme solution
containing RPMI 1640, 5% FCS, penicillin (100 U/mL)/streptomycin
(100 .mu.g/mL) and 0.25 mg/mL Collgenase type VIII (Sigma-Aldrich).
In order to collect the small intestine lamina propria cells,
fragments were vortexed for 10 s after each incubation and poured
over a 70 .mu.m cell strainer. Cell were washed once and used for
flow cytometry.
Flow Cytometry Analysis of T Cell Subsets:
Phenotypic characterisation of T cell subsets was performed by
means of flow cytometry. Cells were resuspended in PBS/1% BSA and
were incubated for 15 min with anti-mouse CD16/CD32 (Mouse BD Fc
Block; BD Pharmingen, San Jose, Calif., USA). For determining the
T.sub.h1/T.sub.h2 subsets, cells were extracellularly stained with
CD4-PerCp-Cy5.5, CD69-APC, CXCR3-PE (eBiosciences, San Diego,
Calif., USA) and T1ST2-FITC (MD Biosciences, St. Paul, Minn., USA).
After staining extracellular markers, cells were stained with a
fixable viability dye AlexaFluor780 (eBioscience). Results were
collected with BD FACSCanto II flow cytometer (Becton Dickinson,
Franklin Lakes, N.J., USA) and were analysed with FlowLogic
software (Inivai Technologies, Mentone, VIC, Australia).
Ex Vivo Re-Stimulation Assay and Cytokine Levels:
After sacrifice, spleens were removed and a single cell suspension
was obtained. Then splenocytes (6.times.10.sup.5 cells) were
cultured either with medium or with 500 .mu.g/mL whey protein at
37.degree. C., 5% CO.sub.2. After 5 days of incubation,
supernatants were collected and stored at -20.degree. C. until
further analysis. Cytokine quantification of IL-5, IL-13, IL-10,
IL-17A and IFN-.gamma. was performed by means of a Cytometric Bead
Array (CBA) Flex Set assay (BD Biosciences) following
manufacturer's instructions. Beads were analysed with BD FACSCanto
II flow cytometer and results were obtained in FCAP v. 3.0 software
(Becton Dickinson).
Statistical Analysis:
All statistical analyses used GraphPad Prism 6.0c software for
Macintosh (GraphPad Software, San Diego, Calif., USA). All data was
analysed for normality and equality of variance. One-way ANOVA,
followed by a Bonferroni's multiple comparison post hoc test for
selected groups (7 pre-selected comparisons) was used when
possible. When data was not normally distributed, as in the case of
cytokines levels, it was first LOG-transformed and tested again. If
LOG transformation did not improve normality, then the
non-parametric Kruskal-Wallis test was used, followed by a Dunn's
post hoc for selected groups and 7 pre-selected comparisons. All
data is presented as mean.+-.SEM of 4-8 animals per group.
P<0.05 was considered of statistical significance.
Results
In order to investigate the local effects in the intestine, lamina
propria lymphocytes from the small intestine (SI-LP) were isolated
and analysed by flow cytometry for the different T cell subsets. In
line with the paradigm that allergy influences the balance between
T.sub.h1 and T.sub.h2 lymphocytes skewing it toward the T.sub.h2
environment (Cox, H E, J Pediatr Gastroenterol Nutr 2008; 47 Suppl
2, S45-48), it was observed that allergic mice showed significantly
lower numbers of activated T.sub.h1 cells, while activated T.sub.h2
cells appeared to increase in number (FIG. 3). As a result, the
ratio of activated T.sub.h1/T.sub.h2 cells was shifted in favour of
T.sub.h2 in the allergic control mice (Group 2), while prior
feeding with peptides and synbiotics (Group 5) prevented this
shift. Feeding mice only the synbiotics-supplemented diet (Group 4)
tended to preserve the T.sub.h1/T.sub.h2 balance but less
pronounced compared to the combination with peptides, suggesting
that the synbiotics ensure a favourable milieu during the
presentation of the peptides by antigen presenting cells.
To investigate whether the preventive treatments affect the
functionality of cells in the systemic compartment, spleens from
the treated groups were collected 18 h after the oral challenge and
splenic lymphocytes were stimulated ex vivo with allergen for 5
days in order to determine their capacity to produce cytokines. The
results presented in FIG. 4 show that the functionality of
splenocytes was affected by pre-exposure to the combination of
peptides and synbiotics-enriched diet (Group 5). Functionality was
assessed by assaying allergy-related T.sub.h2 cytokines (IL-13,
IL-5, IL-10) as well as T.sub.h1-(IFN-.gamma.) and Th17-assosiated
ones (IL-17A). Notably, cells from allergic controls (Group 2)
markedly produced all these cytokines; not only the
T.sub.h2-associated cytokines. However, cells from animals treated
with a combination of peptide mixture and synbiotics (Group 5)
tended to induce less IL-17A, IL-10 and IL-13, as compared to the
peptide mixture alone (Group 3).
These results indicate that combined exposure to peptide mixture
and synbiotics reduces allergen-induced cytokine production and
prevents unfavourable shift in the T.sub.h1/T.sub.h2 balance in the
intestinal lamina propria.
Example 4
Materials and Methods
Peptides:
Same as example 1 except that prior to the animal experiment
peptides were dissolved in PBS and a mixture of all four peptides
was prepared in which each peptide was at a concentration of 0.8
mg/mL ("PepMix")
Animals:
Same as in example 1.
Statistical Analysis:
Statistical analyses were conducted using GraphPad Prism 6.0c
software for Macintosh (GraphPad Software, San Diego, Calif., USA).
Data was analysed with one-way ANOVA and post hoc Dunnett's
multiple comparison test. Data is presented as mean.+-.SEM of 6-8
animals per group. P<0.05 was considered of statistical
significance.
Diets:
Semipurified cow's milk protein-free standard mouse chow was
composed based on AIN-93G recipe (control diet) and supplemented
with 2 wt % 2.times.10.sup.9 CFU/g Bifidobacterium breve M-16V
(Morinaga Milk Industry Co., Ltd, Tokyo, Japan) (probiotic diet).
The probiotic component was mixed through the diet and the mixture
was pressed into pellets. Diets were stored at -4.degree. C. prior
to use.
Study Protocol:
Mice were orally exposed (using a blunt needle) to 0.5 mL of the
PepMix or phosphate buffered saline (PBS, Lonza, Walkerville, Md.,
USA) prior to sensitization (daily; from day -7 to day -2). In the
same week (from day -9 to day 0) mice were fed the control diet or
the probiotic diet ad libitum. Subsequently, on day 0, 7, 14, 21
and 28, mice were sensitized orally with 20 mg whey protein (DMV
International, Veghel, The Netherlands) homogenized in 0.5 mL PBS
and mixed with 10 .mu.g cholera toxin (CT; List Biological
Laboratories, Inc. California, USA) as an adjuvant. The
non-sensitized mice received 10 .mu.g cholera toxin in 0.5 mL PBS
only. Table 3 summarizes the five groups tested.
Five days after the last sensitization, mice underwent an
intradermal whey challenge (injection in the ear pinnae with 10
.mu.g whey protein in 20 .mu.l PBS) and the acute allergic skin
response was recorded, as described in example 1.
TABLE-US-00005 TABLE 3 Interventions in the different groups Group
Pre-treatment Sensitization Challenge 1 negative control PBS +
control diet PBS + CT whey 2 positive control PBS + control diet
whey + CT whey 3 peptide PepMix + control diet whey + CT whey 4
probiotics PBS + probiotic diet whey + CT whey 5 peptide + PepMix +
probiotic whey + CT whey probiotics diet
Results
Skin response results are summarized in FIG. 5. Sensitized, but
untreated animals (Group 2) developed a significantly higher
allergic response compared to the non-sensitised ones (Group 1).
The treatment with probiotic alone (Group 4) did not significantly
reduce the allergic response. However, the allergic response was
significantly reduced after pre-exposing the animals to a mixture
of tolerogenic peptides alone (Group 3) and this reduction was more
pronounced when the tolerogenic peptides were combined with a diet
containing probiotics (Group 5).
SEQUENCE LISTINGS
1
51162PRTBos taurus 1Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile
Gln Lys Val Ala1 5 10 15Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser
Asp Ile Ser Leu Leu 20 25 30Asp Ala Gln Ser Ala Pro Leu Arg Val Tyr
Val Glu Glu Leu Lys Pro 35 40 45Thr Pro Glu Gly Asp Leu Glu Ile Leu
Leu Gln Lys Trp Glu Asn Gly 50 55 60Glu Cys Ala Gln Lys Lys Ile Ile
Ala Glu Lys Thr Lys Ile Pro Ala65 70 75 80Val Phe Lys Ile Asp Ala
Leu Asn Glu Asn Lys Val Leu Val Leu Asp 85 90 95Thr Asp Tyr Lys Lys
Tyr Leu Leu Phe Cys Met Glu Asn Ser Ala Glu 100 105 110Pro Glu Gln
Ser Leu Ala Cys Gln Cys Leu Val Arg Thr Pro Glu Val 115 120 125Asp
Asp Glu Ala Leu Glu Lys Phe Asp Lys Ala Leu Lys Ala Leu Pro 130 135
140Met His Ile Arg Leu Ser Phe Asn Pro Thr Gln Leu Glu Glu Gln
Cys145 150 155 160His Ile218PRTArtificialpeptide derived from cow's
milk beta-lactoglobulin 2Gln Lys Val Ala Gly Thr Trp Tyr Ser Leu
Ala Met Ala Ala Ser Asp1 5 10 15Ile Ser318PRTArtificialpeptide
derived from cow's milk beta-lactoglobulin 3Trp Tyr Ser Leu Ala Met
Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala1 5 10 15Gln
Ser418PRTartificialpeptide derived from cow's milk
beta-lactoglobulin 4Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser
Ala Pro Leu Arg1 5 10 15Val Tyr518PRTartificialpeptide derived from
cow's milk beta-lactoglobulin 5Leu Leu Asp Ala Gln Ser Ala Pro Leu
Arg Val Tyr Val Glu Glu Leu1 5 10 15Lys Pro
* * * * *